Conformational dynamics of the ClpA and ClpP oligomers. (GP, AG, MRM)[unreadable] [unreadable] ClpA, a Hsp100/Clp chaperone from E. Coli, forms a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. The precise mechanism of ATP driven unfoldase activity of ClpA and pathway for substrate fragments discharge from the ClpAP complexes are important but still not well understood problems. To gain insight into those processes we have used sedimentation velocity measurements to determine overall conformational changes of the ClpA hexamer upon peptide binding and in different nucleotide states. The ClpP tetradecamer stability and ClpAP complexes have also been studied by the same method. Previous calorimetric titrations of ClpA hexamer with SsrA target peptide have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. This observations were confirmed by analytical ultracentrifugation studies indicating that ClpA hexamer conformational changes induced by SsrA peptide binding are reflected by sedimentation coefficient distributions.[unreadable] [unreadable] ClpA has two ATP binding domains (D1 and D2) and both the enzyme oligomerization and unfoldase activity is nucleotide dependent. In previous experiments ATPgS, the nonhydrolyzable ATP analog have been used. To study conformational changes of ClpA in different nucleotide states single point mutations in ClpA nucleotide binding Walker-B motifs of domains D1 and D2 were introduced. The double D1/D2 ClpA mutant forms stable hexamers in the presence of ATP, as confirmed by analytical ultracentrifugation, but has no detectable ATPase activity.[unreadable] [unreadable] The oligomerization studies of ClpP protease form E. Coli indicate that ClpP tetradecamers disassemble at very low protein concentration, which may facilitate removal of degradation fragments from the ClpP proteolytic chamber and may also play a role in macromolecular dynamics of ClpAP complexes. To study the importance of this process a crosslinked cysteine mutant of ClpP was used. As confirmed by analytical ultracentrifugation, crosslinked ClpP maintains the tetradecamer form even at extremely low protein concentrations. This ClpP mutant will provide an important tool to study the dynamics of ClpAP complexes.[unreadable] [unreadable] Oxidation dependent oligomerization of the human Peroxiredoxin I (hPrxI). (GP, AG, DYL, SGR)[unreadable] [unreadable] Human Peroxiredoxin I belongs to a large and diverse family of antioxidant enzymes that also regulate cell signaling pathways, apoptosis and differentiation. Peroxiredoxins (Prxs) can be regulated by changes to phosphorylation and redox state and oligomerization is believed to play a key role in this regulation. Most of 2-Cys Prxs demonstrate large changes in their oligomeric state, forming either dimers or decamers with the characteristic doughnut shape. Prxs oligomerization is promoted by their oxidation state but also by other factors, such as ionic strength and pH.[unreadable] [unreadable] Oligomerization of human Prx I was studied by analytical ultracentrifugation. Since hPrx I can be reversibly hyperoxidized to cysteinesulfinic acid (SO2H), both reduced, oxidized and hyperoxidized forms of this enzyme were characterized using large range of protein concentrations and different buffer conditions. Sedimentation velocity experiment show that hPrxI redox-dependent oligomerization is strikingly different from other Prxs, reported in the literature. The oxidized hPrxI is an obligate homodimer and the reduced form of this enzyme is in a concentration dependent monomer-dimer-decamer equilibrium. The hyperoxidized form of hPrxI also forms decamers, and the concentration dependence of this process in higher than that of the reduced form of the enzyme. The pH and ionic strength dependence of hPrxI oligomerization was studied in a range of buffer conditions. Interaction of different forms of hPrxI with human Sulfiredoxin (hSrx) was also examined.[unreadable] [unreadable] Capping Protein - CARMIL Interaction Studies. (GP, AG, TU, JAH)[unreadable] [unreadable] Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics. CP binds to the barbed end of the actin filament blocking both association and dissociation of actin monomers. One potential regulator of CP is CARMIL, that might act as potent CP antagonist and inhibit CP interaction with actin filaments.[unreadable] [unreadable] Two regions on of the CARMIL protein (CAH3-a and CAH3-b) were identified as crucial for interactions with CP. Cloned fragments of CARMIL with sequences encompassing both the CAH3-a and CAH3-b regions, with several single point mutations with either of them, were used to study CARMIL interaction with mouse Capping Protein (mCP) heterodimer (a, b) by Isothermal Titration Calorimetry (ITC). Results obtained by ITC were compared with those from an actin polymerization inhibition assay.[unreadable] [unreadable] The C-terminals of CP a and b subunits form tentacles that can make extensive contacts with actin when CP binds to the barbed end of the filament. The mCP deletion mutant lacking the tentacle on the a subunit (mCPa1D) is unable to make tight complexes with actin. Since CARMIL may potentially regulate capping by interactions with C-terminals of mCP a and b subunits, both mCPa1D and mCPb2D (lacking the tentacle on the b subunit) were used to study their interactions with several CARMIL CAH3 mutants by ITC.